Patent application title: LOADABLE POLYMERIC PARTICLES FOR BONE AUGMENTATION AND METHODS OF PREPARING AND USING THE SAME

Abstract:

Particles are provided for use in therapeutic and/or diagnostic
procedures. The particles include poly[bis(trifluoroethoxy)phosphazene]
and/or a derivatives thereof which may be present throughout the
particles or within an outer coating of the particles. The particles may
also include a core having a hydrogel formed from an acrylic-based
polymer. Such particles may be provided for placement within defects in
bone within the body of a mammal to augment structural support and
facilitate osteogenesis without causing adverse reactions therein. The
hydrogel core may further be used as a delivery vehicle for therapeutic
agents to treat or retard pathologic processes within the bone defect
during healing.

Claims:

1. A particle for augmentation of a mammalian bone defect, the particle
comprising a core and a coating, wherein:the core comprises a hydrogel, a
ceramic, a metal, an alloy, a metal compound, a polymer, or any
combination thereof; andthe coating comprises a polyphosphazene having
the formula: ##STR00002## whereinn is 2 to ∞; andR1 to R6
are each selected independently from alkyl, aminoalkyl, haloalkyl,
thioalkyl, thioaryl, alkoxy, haloalkoxy, aryloxy, haloaryloxy,
alkylthiolate, arylthiolate, alkylsulphonyl, alkylamino, dialkylamino,
heterocycloalkyl comprising one or more heteroatoms selected from
nitrogen, oxygen, sulfur, phosphorus, or a combination thereof, or
heteroaryl comprising one or more heteroatoms selected from nitrogen,
oxygen, sulfur, phosphorus, or a combination thereof.

6. The particle of claim 1, wherein the particle is bioabsorbable or
nonbioabsorbable and wherein the particle is provided as a sphere or a
microsphere.

7. The particle of claim 1, wherein the particle is provided in a film, a
suspension, a gel, or a paste.

8. The particle of claim 1, wherein the core comprises one or more active
agents.

9. The particle of claim 8, wherein the active agent is a steroid, a
hormone, a nucleic acid, an antibiotic, an antiseptic, an
osteogenesis-enhancing agent, an analgesic, an anti-neoplastic, an
anesthetic, or a biological agent to promote re-growth of bone within the
defects.

10. The particle of claim 8, wherein the active agent is selected from a
bone adhesive, a biocompatible tissue glue, a cement, or an adhesive.

11. The particle of claim 9, wherein the active agent is bioabsorbable or
nonbioabsorbable.

12. The particle of claim 1, further comprising an organic or an inorganic
filler.

13. The particle of claim 12, wherein the core is a hydrogel and the
filler is incorporated in the hydrogel core.

14. The particle of claim 12, wherein the filler is a component of an
injection/delivery medium that delivers the particles to the defect.

15. The particles of claim 12, wherein the inorganic filler is
hydroxyapatite.

16. A method of treating a defect in mammalian bone in vivo, comprising:a.
identifying a defect in mammalian bone to be treated;b. placing a hollow
cannula into the defect;c. injecting particles comprising a core and a
coating into the defect; andd. removing the cannula from the
defect;wherein:the core comprises a hydrogel, a ceramic, a metal, an
alloy, a metal compound, a polymer, or any combination thereof; andthe
coating comprises a polyphosphazene having the formula: ##STR00003##
whereinn is 2 to ∞; andR1 to R6 are each selected
independently from alkyl, aminoalkyl, haloalkyl, thioalkyl, thioaryl,
alkoxy, haloalkoxy, aryloxy, haloaryloxy, alkylthiolate, arylthiolate,
alkylsulphonyl, alkylamino, dialkylamino, heterocycloalkyl comprising one
or more heteroatoms selected from nitrogen, oxygen, sulfur, phosphorus,
or a combination thereof, or heteroaryl comprising one or more
heteroatoms selected from nitrogen, oxygen, sulfur, phosphorus, or a
combination thereof.

18. The method according to claim 16, wherein the polyphosphazene is
poly[bis(2,2,2-trifluoroethoxy)]phosphazene or a derivative of
poly[bis(2,2,2-trifluoroethoxy)]phosphazene.

19. The method of claim 16, wherein the core further comprises one or more
active agents.

20. The method of claim 19, wherein the active agent is a steroid, a
hormone, a nucleic acid, an antibiotic, an antiseptic, an
osteogenesis-enhancing agent, an analgesic, an anti-neoplastic, an
anesthetic, a biological agent to promote re-growth of bone within the
defects, a bone adhesive, a biocompatible tissue glue, a cement, an
adhesive, or a combination thereof.

21. The method of claim 16, wherein the particle is bioabsorbable or
nonbioabsorbable and wherein the particle is provided as a sphere or a
microsphere.

22. The method of claim 16, wherein the particle further comprises an
organic or an inorganic filler.

23. The method of claim 16, wherein the core is a hydrogel and the filler
is incorporated in the hydrogel core.

24. A method of delivering an active agent to a defect in mammalian bone
in vivo, the method comprising:a. providing one or more particles, the
particles each comprising a core and a coating, wherein the coating
comprises poly[bis(trifluoroethoxy)phosphazene] and/or a derivative
thereof and an active agent;wherein the core comprises a hydrogel, a
ceramic, a metal, an alloy, a metal compound, a polymer, or any
combination thereof;b. selecting a defect in mammalian bone to be treated
with the active agent; andc. contacting the defect with the one or more
particles such that the active agent is exposed to the localized area.

25. The method of claim 24, wherein the active agent is a steroid, a
hormone, a nucleic acid, an antibiotic, an antiseptic, an
osteogenesis-enhancing agent, an analgesic, an anti-neoplastic, an
anesthetic, a biological agent to promote re-growth of bone within the
defect, a bone adhesive, a biocompatible tissue glue, a cement, or an
adhesive.

Description:

BACKGROUND OF THE INVENTION

[0001]Small particles, including microspheres and nanospheres, have many
medical uses in diagnostic and therapeutic procedures. In selected
clinical applications, it may be advantageous to deliver bioabsorbable
microspheres to an affected bone defect or cavity within the body of a
mammal to provide a non-permanent bone anchoring substrate to augment
missing bone and enable faster osteogenesis and regeneration of natural
bone tissue without causing adverse reactions therein.

[0002]Most particles used in medical applications are characterized by
numerous disadvantages including irritation of the tissues with which
they come in contact and initiation of adverse immune reactions.
Additionally, many of the materials used to prepare these particles may
degrade relatively rapidly within the mammalian body, thereby detracting
from their utility in certain procedures where long term presence of
intact particles may be necessary. Moreover, the degradation of materials
may release toxic or irritating compounds causing adverse reactions in
the patients.

[0003]Some known particle types suffer from difficulties in achieving
desirable suspension properties when the particles are incorporated into
a delivery suspension for injection into a site in the body to be
treated. Many times, the particles settle out or tend to "float" in the
solution such that they are not uniformly suspended for even delivery.
Furthermore, particles may tend to aggregate within the delivery solution
and/or adhere to some part of the delivery device, making it necessary to
compensate for these adhesive/attractive forces.

[0004]In order to achieve a stable dispersion, suitable dispersing agents
may be added, which may include surfactants directed at breaking don
attractive particle interactions. Depending on the nature of the particle
interaction, materials such as the following may be used: cationic,
anionic or nonionic surfactants such as Tween® 20, Tween® 40,
Tween® 80, polyethylene glycols, sodium dodecyl sulfate, various
naturally occurring proteins such as serum albumin, or any other
macromolecular surfactants in the delivery formulation. Furthermore
thickening agents can be used help prevent particles from settling by
sedimentation and to increase solution viscosity, for example, polyvinyl
alcohols, polyvinyl pyrrolidones, sugars or dextrins. Density additives
may also be used to achieve buoyancy.

[0005]It can also be difficult to visualize microparticles in solution to
determine their degree of suspension when using clear, transparent
polymeric acrylate hydrogel beads in aqueous suspension. The inert
precipitate barium sulfate, may be used in particle form as an additive
for bone cement, for silicones for rendering items visible during X-ray
examination and for providing radiopacity to polymeric acrylate
particles. See Jayakrishnan et al., Bull. Mat. Sci., Vol. 12, No. 1, pp.
17-25 (1989). Barium sulfate also is known for improving fluidization,
and is often used as an inorganic filler to impart anti-stick behavior to
moist, aggregated particles. Other prior art attempts to increase
visualization of microparticles include the use of gold, for example, in
Embosphere Gold®, which provides a magenta color to acrylate
microparticles using small amounts of gold.

[0006]In certain medical applications, it may be of farther value to
provide microparticles such as microspheres in one or more sizes.
Furthermore, it may also be of value to provide each of such sizes of
microspheres incorporated with color-coded associated dyes to indicate
the microsphere size to the user. In yet other applications of use, it
may further be of value to provide sized and color-coded microspheres to
a user in similarly color-coded syringes or other containers for
transport and delivery to further aid a user in identifying the size of
microspheres being used.

[0009]The glycoprotein Erythropoietin (EPO) regulates the production of
red blood cells by its specific interaction with the cell-surface
receptor EPOR (see: Krantz S B. Erythropoietin. Blood 1999; 77: 419-34).
Additionally, EPOR is expressed in several nonhematopoietic cell types
(see: D'Andrea A D, Lodish H F, Wong G G. Expression cloning of the
murine erythropoietin receptor. Cell 1989; 57: 277-85). For instance,
studies have shown that, n the brain EPO-EPOR signalling is associated
with the response to neuronal injury. In the kidney, the intestine and in
muscle cells, EPO has been shown to induce cellular proliferation. EPOR
was also detected in several types of vascular endothelial cells. Recent
studies have further demonstrated that EPO is able to promote
angiogenesis (s3e: Folkman J, Shing Y: Angiogenesis. J Biol Chem 1992;
267: 10931-4).

[0010]The cytokine VEGF shares significant homology with EPO. Both, the
expression of EPO and VEGF are stimulated by hypoxia through an
analogical pathway. Simultaneously, hypoxia and oxygen tension play a
crucial role in the process of fracture healing. As above-mentioned, EPO
and VEGF have also been shown to promote angiogenesis and cell
proliferation. In addition, the VEGF gene and the EPO gene have
substantial similarities in terms of structure and enhancer elements.

[0011]Thus, there exists in the art a need for small particles that can be
formed to have a preferential generally spherical configuration which are
not degraded by the natural systems of the mammalian system, are
biocompatible, are easy to visualize in suspension while in use and/or
demonstrate acceptable physical and suspension properties for certain
applications such as various therapeutic procedures involving bone
injuries or diseases resulting in bone defects in mammals.

BRIEF SUMMARY OF THE INVENTION

[0012]The invention includes a particle for use in a therapeutic and/or
diagnostic procedure in which a plurality of the particles is injected or
otherwise introduced into a bone defect or cavity within the body of a
mammal to augment missing bone and facilitate bone regrowth and healing
therein. The particle comprises poly[bis(trifluoroethoxy)phosphazene]
and/or a derivative thereof. Poly[(bistrifluorethoxy)phosphazene has
antibacterial properties and inhibits the accumulation of thrombocytes.
Particles comprising poly[bis(trifluoroethoxy)phosphazene] can be formed
to have a generally spherical configuration and are biocompatible and
easy to visualize.

[0013]The present invention further includes particles comprising
poly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof
provided as microspheres provided in one or more specified sizes.

[0014]Further described herein is a method of delivering an active agent
to a localized area involving a bone defect within a body of a mammal
comprising contacting the localized area with at least one of a particle
comprising poly[bis(trifluoroethoxy)phosphazene] and/or a derivative
thereof and an active agent, such that an effective amount of the active
agent is exposed to the localized area.

[0015]The invention also includes a method of delivering an active agent
to a localized area within the body of a mammal comprising contacting the
localized area with a plurality of particles comprising
poly[bis(trifluoroethoxy)phosphazene] and/or a derivative thereof. The
particles may further comprise one or more active agents, the active
agent(s) may act to retard infection, inflammation, pain, other
pathologic conditions, and/or add structural strength and promote
osteogenesis in the bone defect.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0016]The foregoing summary, as well as the following detailed description
of the invention, will be better understood when read in conjunction with
the appended drawings. For the purpose of illustrating the invention,
there are shown in the drawings embodiments that are presently preferred.
It should be understood, however, that the invention is not limited to
the precise arrangements and instrumentalities shown.

[0017]In the drawings:

[0018]FIG. 1 shows a schematic representation of a general cryoextraction
scheme used to prepare particles according to one embodiment of the
invention;

[0019]FIG. 2 shows the manual dripping technique by which the polymer
solution was supplied to liquid nitrogen in preparation of the
microspheres of Example 1, herein;

[0020]FIG. 3A and FIG. 3B show unloaded polyphosphazene particles
(microspheres) as prepared by one embodiment of the cryoextraction method
as described herein. FIG. 3A shows a 4× optical microscope view and
FIG. 3B shows a 100× scanning electron microscope view;

[0021]FIG. 4 shows a particle (microsphere) formed according to one
embodiment of the invention loaded with bovine insulin (20% (wt/wt)) at
100× magnification SEM;

[0022]FIG. 5A and FIG. 5B show the surface morphology of unloaded
polyphosphazene microspheres. FIG. 5A is an image obtained using an
atomic force microscope and FIG. 5B is a scanning electron micrograph
showing the surface of an unloaded polyphosphazene microsphere at
5000× magnification;

[0023]FIGS. 6 and 7 show a cryoextraction setup for use in an embodiment
of the invention wherein FIG. 6 is a cryoextraction vessel and FIG. 7 is
a syringe pump;

[0024]FIG. 8 is a cross-sectional view of an apparatus for use in
microcatheter testing of microparticles in Example 14 herein;

[0025]FIGS. 9A and 9B show an SEM at 1.0K× magnification of the
surface of the Sample C microparticles just after the
hydration/dehydration cycle and at a 50.00K× magnification of the
film thickness of microparticles formed in accordance with Sample C of
Example 12 used in the evaluation of Example 14, respectively;

[0026]FIGS. 10A, 10B, 10C and 10D are SEMs of microparticles made in
accordance with Sample C of Example 12 used in the evaluation of Example
14 after passing through a catheter showing surface features (FIGS. 10A,
10B and 10C) at 1.0K× magnification and at 5.0K×
magnification (FIG. 10D); and

[0027]FIGS. 11A, 11B, 11C and 11D are SEMs of microparticles formed in
accordance with Sample C of Example 12 after thermal stress testing in
Example 14. FIG. 11A is a 50× magnification of a minor amount of
delamination in the strong white contrast portion. FIG. 11B is a
200× magnification of the microparticles of FIG. 11A. FIGS. 11C and
11D are, respectively, 200× and 1.0K× magnified SEMs of other
Sample C microparticles showing only minor defects.

[0028]FIGS. 12A and B show the injection of exemplary microspheres of the
present invention into a bone defect to augment missing bone and
facilitate healing therein.

[0030]FIG. 14. illustrates better ingrowth stability of
poly[bis(trifluoroethoxy)phosphazene] coated implants after 6 weeks
compared to the control group, as measured by the implant bone relative
movement with a 3000 g strain. (1 is Group 1a, 2 is Group 1b, 3 is group
2a, and 4 is Group 2b).

[0031]FIG. 15. illustrates a histological figure of an implanted cylinder.

DETAILED DESCRIPTION OF THE INVENTION

[0032]Described herein are particles that may be manufactured using
poly[bis(trifluoroethoxy)phosphazene] and/or derivatives thereof as well
as methods of preparing such particles. Additionally, described herein
are therapeutic and/or diagnostic methods and procedures which use the
particles as described herein, including methods of treating bone defects
in mammalian bone to facilitate osteogenesis and bone healing.

[0033]Also included are sustained release drug delivery formulations for
oral administration including the particles for localized delivery of an
active agent to the gastrointestinal system and/or systemic delivery of
an active agent as well as a sustained release drug delivery formulation
that can be injected subcutaneously or intravenously for localized
delivery of an active agent.

[0034]All of the methods, compositions and formulations of the invention
utilize at least one particle as described herein. "Particle" and
"particles" as used herein mean a substantially spherical or ellipsoid
article(s), hollow or solid, that may have any diameter suitable for use
in the specific methods and applications described below, including a
microparticle(s), a microsphere(s) and a nanosphere(s), beads and other
bodies of a similar nature known in the art.

[0035]The preferred particles of the invention according to one embodiment
described herein are composed, in whole or in part, the specific
polyphosphazene polymer known as poly[bis(trifluoroethoxy)phosphazene] or
a derivative of poly[bis(trifluoroethoxy)phosphazene]. Use of this
specific polymer provides particles that are at least in part inorganic
in that they include an inorganic polymer backbone and which are also
biocompatible in that when introduced into a mammal (including humans and
animals), they do not significantly induce a response of the specific or
non-specific immune systems. The scope of the invention also includes the
use(s) of such particles as controlled drug delivery vehicles.

[0036]The particles are useful in a variety of therapeutic and/or
diagnostic procedures in part because, owing to the biocompatible nature
of the polymer, the particles facilitate avoidance or elimination of
immunogenic reactions generally encountered when foreign bodies are
introduced into a mammalian body, such as "implant rejection" or
"allergic shock," and other adverse reactions of the immune system.
Moreover, it has been found that the particles of the invention exhibit
reduced biodegradation in vivo, thereby increasing the long-term
stability of the particle in the biological environment. Moreover, in
those situations where some degradation is undergone by the polymer in
the particle, the products released from the degradation include only
non-toxic concentrations of phosphorous, ammonia, and trifluoroethanol,
which, advantageously, is known to promote anti-inflammatory responses
when in contact with mammalian tissue.

[0037]Each of the particles in the invention is formed at least in part of
the polymer, poly[bis(2,2,2-trifluoroethoxy)phosphazene] or a derivative
thereof (referred to further herein as
"poly[bis(trifluoroethoxy)phosphazene]". As described herein, the polymer
poly[bis(2,2,2-trifluoroethoxy)phosphazene] or derivatives thereof have
chemical and biological qualities that distinguish this polymer from
other know polymers in general, and from other know polyphosphazenes in
particular. In one aspect of this invention, the polyphosphazene is
poly[bis(2,2,2-trifluoroethoxy)phosphazene] or derivatives thereof such
as other alkoxide, halogenated alkoxide, or fluorinated alkoxide
substituted analogs thereof. The preferred
poly[bis(trifluoroethoxy)phosphazene] polymer is made up of repeating
monomers represented by the formula (I) shown below:

##STR00001##

wherein R1 to R6 are all trifluoroethoxy (OCH2CF3)
groups, and wherein n may vary from at least about 40 to about 100,000,
as disclosed herein. Alternatively, one may use derivatives of this
polymer in the present invention. The term "derivative" or "derivatives"
is meant to refer to polymers made up of monomers having the structure of
formula I but where one or more of the R1 to R6 functional
group(s) is replaced by a different functional group(s), such as an
unsubstituted alkoxide, a halogenated alkoxide, a fluorinated alkoxide,
or any combination thereof, or where one or more of the R1 to
R6 is replaced by any of the other functional group(s) disclosed
herein, but where the biological inertness of the polymer is not
substantially altered.

[0038]In one aspect of the polyphosphazene of formula (I) illustrated
above, for example, at least one of the substituents R1 to R6
can be an unsubstituted alkoxy substituent, such as methoxy (OCH3),
ethoxy (OCH2CH3) or n-propoxy (OCH2CH2CH3). In
another aspect, for example, at least one of the substituents R1 to
R6 is an alkoxy group substituted with at least one fluorine atom.
Examples of useful fluorine-substituted alkoxy groups R1 to R6
include, but are not limited to OCF3, OCH2CF3,
OCH2CH2CF3, OCH2CF2CF3,
OCH(CF3)2, OCCH3(CF3)2,
OCH2CF2CF2CF3, OCH2(CF2)3CF3,
OCH2(CF2)4CF3, OCH2(CF2)CF3,
OCH2(CF2)6CF3, OCH2(CF2)7CF3,
OCH2CF2CHF2, OCH2CF2CF2CHF2,
OCH2(CF2)3CHF2, OCH2(CF2)4CHF2,
OCH2(CF2)5CHF2, OCH2(CF2)6CHF2,
OCH2(CF2)7CHF2, and the like. Thus, while
trifluoroethoxy (OCH2CF3) groups are preferred, these further
exemplary functional groups also may be used alone, in combination with
trifluoroethoxy, or in combination with each other. In one aspect,
examples of especially useful fluorinated alkoxide functional groups that
may be used include, but are not limited to,
2,2,3,3,3-pentafluoropropyloxy (OCH2CF2CF3),
2,2,2,2',2',2'-hexafluoroisopropyloxy (OCH(CF3)2),
2,2,3,3,4,4,4-heptafluorobutyloxy (OCH2CF2CF2CF3),
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyloxy
(OCH2(CF2)7CF3), 2,2,3,3,-tetrafluoropropyloxy
(OCH2CF2CHF2), 2,2,3,3,4,4-hexafluorobutyloxy
(OCH2CF2CF2CHF2),
3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorooctyloxy
(OCH2(CF2)7CHF2), and the like, including
combinations thereof.

[0039]Further, in some embodiments, 1% or less of the R1 to R6
groups may be alkenoxy groups, a feature that may assist in crosslinking
to provide a more elastomeric phosphazene polymer. In this aspect,
alkenoxy groups include, but are not limited to,
OCH2CH═CH2, OCH2CH2CH═CH2, allylphenoxy
groups, and the like, including combinations thereof. Also in formula (I)
illustrated herein, the residues R1 to R6 are each
independently variable and therefore can be the same or different.

[0040]By indicating that n can be as large as ∞ in formula I, it is
intended to specify values of n that encompass polyphosphazene polymers
that can have an average molecular weight of up to about 75 million
Daltons. For example, in one aspect, n can vary from at least about 40 to
about 100,000. In another aspect, by indicating that n can be as large as
∞ in formula I, it is intended to specify values of n from about
4,000 to about 50,000, more preferably, n is about 7,000 to about 40,000
and most preferably n is about 13,000 to about 30,000.

[0041]In another aspect of this invention, the polymer used to prepare the
polymers disclosed herein has a molecular weight based on the above
formula, which can be a molecular weight of at least about 70,000 g/mol,
more preferably at least about 1,000,000 g/mol, and still more preferably
a molecular weight of at least about 3×106 g/mol to about
20×106 g/mol. Most preferred are polymers having molecular
weights of at least about 10,000.000 g/mol.

[0042]In a further aspect of the polyphosphazene formula (I) illustrated
herein, n is 2 to ∞, and R1 to R6 are groups which are
each selected independently from alkyl, aminoalkyl, haloalkyl, thioalkyl,
thioaryl, alkoxy, haloalkoxy, aryloxy, haloaryloxy, alkylthiolate,
arylthiolate, alkylsulphonyl, alkylamino, dialkylamino, heterocycloalkyl
comprising one or more heteroatoms selected from nitrogen, oxygen,
sulfur, phosphorus, or a combination thereof, or heteroaryl comprising
one or more heteroatoms selected from nitrogen, oxygen, sulfur,
phosphorus, or a combination thereof. In this aspect of formula (I), the
pendant side groups or moieties (also termed "residues") R1 to
R6 are each independently variable and therefore can be the same or
different. Further, R1 to R6 can be substituted or
unsubstituted. The alkyl groups or moieties within the alkoxy,
alkylsulphonyl, dialkylamino, and other alkyl-containing groups can be,
for example, straight or branched chain alkyl groups having from 1 to 20
carbon atoms, typically from 1 to 12 carbon atoms, it being possible for
the alkyl groups to be further substituted, for example, by at least one
halogen atom, such as a fluorine atom or other functional group such as
those noted for the R1 to R6 groups above. By specifying alkyl
groups such as propyl or butyl, it is intended to encompass any isomer of
the particular alkyl group.

[0043]In one aspect, examples of alkoxy groups include, but are not
limited to, methoxy, ethoxy, propoxy, and butoxy groups, and the like,
which can also be further substituted. For example the alkoxy group can
be substituted by at least one fluorine atom, with 2,2,2-trifluoroethoxy
constituting a useful alkoxy group. In another aspect, one or more of the
alkoxy groups contains at least one fluorine atom. Further, the alkoxy
group can contain at least two fluorine atoms or the alkoxy group can
contain three fluorine atoms. For example, the polyphosphazene that is
combined with the silicone can be
poly[bis(2,2,2-trifluoroethoxy)phosphazene]. Alkoxy groups of the polymer
can also be combinations of the aforementioned embodiments wherein one or
more fluorine atoms are present on the polyphosphazene in combination
with other groups or atoms.

[0044]Examples of alkylsulphonyl substituents include, but are not limited
to, methylsulphonyl, ethylsulphonyl, propylsulphonyl, and butylsulphonyl
groups. Examples of dialkylamino substituents include, but are not
limited to, dimethyl-, diethyl-, dipropyl-, and dibutylamino groups.
Again, by specifying alkyl groups such as propyl or butyl, it is intended
to encompass any isomer of the particular alkyl group.

[0045]Exemplary aryloxy groups include, for example, compounds having one
or more aromatic ring systems having at least one oxygen atom,
non-oxygenated atom, and/or rings having alkoxy substituents, it being
possible for the aryl group to be substituted for example by at least one
alkyl or alkoxy substituent defined above. Examples of aryloxy groups
include, but are not limited to, phenoxy and naphthoxy groups, and
derivatives thereof including, for example, substituted phenoxy and
naphthoxy groups.

[0046]The heterocycloalkyl group can be, for example, a ring system which
contains from 3 to 10 atoms, at least one ring atom being a nitrogen,
oxygen, sulfur, phosphorus, or any combination of these heteroatoms. The
hetereocycloalkyl group can be substituted, for example, by at least one
alkyl or alkoxy substituent as defined above. Examples of
heterocycloalkyl groups include, but are not limited to, piperidinyl,
piperazinyl, pyrrolidinyl, and morpholinyl groups, and substituted
analogs thereof.

[0047]The heteroaryl group can be, for example, a compound having one or
more aromatic ring systems, at least one ring atom being a nitrogen, an
oxygen, a sulfur, a phosphorus, or any combination of these heteroatoms.
The heteroaryl group can be substituted for example by at least one alkyl
or alkoxy substituent defined above. Examples of heteroaryl groups
include, but are not limited to, imidazolyl, thiophene, furane, oxazolyl,
pyrrolyl, pyridinyl, pyridinolyl, isoquinolinyl, and quinolinyl groups,
and derivatives thereof, such as substituted groups.

[0048]The diameter of a particle formed according to the invention will
vary depending on the end application in which the particle is to be
used. The diameter of such particles is preferably about 1 to about 5,000
μm, with a diameter of about 1 to about 1,000 μm being most
preferred. Other preferred sizes include diameters of about 200 to about
500 μm, about 1 to about 200 μm and greater than about 500 μm.
In methods using the particle where more than one particle is preferred
it is not necessary that all particles are of the same diameter or shape.

[0049]The particles may also include other compounds which function to
enhance, alter or otherwise modify the behavior of the polymer or
particle either during its preparation or in its therapeutic and/or
diagnostic use. For example, active agents such as peptides, proteins,
hormones, carbohydrates, polysaccharides, nucleic acids, lipids,
vitamins, steroids and organic or inorganic drugs may be incorporated
into the particle. Excipients such as dextran, other sugars, polyethylene
glycol, glucose, and various salts, including, for example, chitosan
glutamate, may be included in the particle.

[0050]Additionally, if desired, polymers other than the
poly[bis(trifluoroethoxy)phosphazene] and/or its derivative may be
included with in the particle. Examples of polymers may include
poly(lactic acid), poly(lactic-co-glycolic acid), poly(caprolactone),
polycarbonates, polyamides, polyanhydrides, polyamino acids,
polyorthoesters, polyacetals, polycyanoacrylates, and polyurethanes.
Other polymers include polyacrylates, ethylene-vinyl acetate co-polymers,
acyl substituted cellulose acetates and derivatives thereof, degradable
or non-degradable polyurethanes, polystyrenes, polyvinylchloride,
polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolefins,
and polyethylene oxide. Examples of polyacrylates include, but are not
limited to, acrylic acid, butyl acrylate, 2-ethylhexyl acrylate, methyl
acrylate, ethyl acrylate, acrylonitrile, methyl methacrylate, TMPTA
(trimethylolpropane triacrylate), and the like. One may incorporate the
selected compounds by any means known in the art, including diffusing,
inserting or entrapping the additional compounds in the matrix of an
already formed particle or by adding the additional compound to a polymer
melt or to a polymer solvent in the preparation of the particle such as
described herein. In a further aspect, for example, the core material can
be selected from among these polymers.

[0051]The loaded or unloaded particle may be coated with an additional
polymer layer or layers, including polymers such as those mentioned
hereinabove. Further, poly[bis(trifluoroethoxy)phosphazene] or its
derivatives may be used to form such a coating on a particle formed of
other suitable polymers or copolymers known or to be developed in the art
that are used to form particles as described herein. Preferably, when
coating a particle such as a microparticle, poly[bis
(trifluoroethoxy)phosphazene is applied as a coating on a
microparticle(s) formed of an acrylic-based polymer as set forth in
further detail below.

[0052]The microspheres may be prepared by any means known in the art that
is suitable for the preparation of particles containing
poly[bis(trifluoroethoxy)phosphazene]. In a procedure according to an
embodiment herein a "polymer solution" is prepared by mixing one or more
polymer solvent(s) and the poly[bis(trifluoroethoxy)phosphazene and/or a
derivative thereof until the polymer is dissolved.

[0053]Suitable solvents for use in the preparation of the polymer solution
include any in which the polymer poly[bis(trifluoroethoxy)phosphazene
and/or its derivatives are soluble. Exemplary solvents include, without
limitation, ethyl-, propyl-, butyl-, pentyl-, octylacetate, acetone,
methylethylketone, methylpropylketone, methylisobutylketone,
tetrahydrofurane, cyclohexanone, dimethylacetamide, acetonitrile,
dimethyl ether, hexafluorobenzene or combinations thereof.

[0054]The polymer solution contains the
poly[bis(trifluoroethoxy)phosphazene and/or its derivative polymer in a
concentration of about 1% by weight of polymer to 20% by weight of
polymer, preferably about 5% to 10% by weight of polymer. Other polymers,
as discussed above, may be present in the solution, or may be added to
the vessel in the form of a second solution powder or other form, if one
wishes to include such polymers in the final particle.

[0055]In carrying out the process, the polymer solution is next dispensed,
preferably in the form of drops or an aerosol, into a vessel containing a
non-solvent. By "non-solvent" it is meant any organic or inorganic
solvents that do not substantially dissolve the
poly[bis(trifluoroethoxy)phosphazene polymer and which have a melting
point that is lower relative to the melting point of the solvent in which
the polymer is dissolved ("polymer solvent"), so that the non-solvent
thaws before the solvent thaws in the course of the incubation step.
Preferably, this difference between the melting point of the non-solvent
and the polymer solvent is about 10° C., more preferably about
15° C., and most preferably, greater than about 20° C.
Under certain conditions it has been found that the structural integrity
of the resultant particle may be enhanced if the difference of the
melting points of the polymer solvent and of the non-solvent is greater
than 15° C. However, it is sufficient that the non-solvent point
is merely slightly lower than that of the polymer solvent.

[0056]The non-solvent/polymer solvent combination is incubated for
approximately 1 to 5 days or until the polymer solvent has been
completely removed from the particles. While not wishing to be bound by
theory, it is hypothesized that during the incubation, the non-solvent
functions to extract the polymer solvent from the microscopic polymer
solution droplets from the particles such that the polymer is at least
gelled. As the incubation period passes, the droplets will shrink and the
solvent becomes further extracted, leading to a hardened outer polymeric
shell containing a gelled polymer core, and finally, after completion of
the incubation, a complete removal of the residual solvent. To ensure
that the polymeric droplets retain a substantially spherical shape during
the incubation period, they may be maintained in a frozen or
substantially gelled state during most if not all of the incubation
period. Therefore, the non-solvent temperature may stay below the melting
point of the solvent during the cryoextraction process.

[0057]As shown in FIG. 1, at the vessel labeled (a), polymer solution
droplets are shown being dispensed either with a syringe or other device
at a controlled rate onto a top layer of liquid nitrogen. The nitrogen
layer is situated over a bottom layer consisting of the selected
non-solvent, which will eventually serve to extract the solvent from the
frozen polymer solution droplets. The non-solvent layer has been
previously frozen with liquid nitrogen prior to the dispensing of the
polymer solution. The vessel labeled (b) shows the onset of the dewing of
the frozen nonsolvent, into which the frozen polymeric droplets will
sink. The vessel labeled (c) shows the cryoextraction procedure after
approximately three days of incubation wherein the polymer solution
droplets, incubated within the non-solvent, have been depleted of a
substantial amount of solvent. The result is a gelled, polymeric particle
in the form of a bead having a hardened outer shell. As can be seen by
the representation, the non-solvent height within the vessel is slightly
reduced due to some evaporation of the non-solvent. The size of the beads
will shrink quite substantially during this process depending on the
initial concentration of the polymer in the polymer solution.

[0058]In one embodiment of a method of preparing a
poly[bis(trifluoroethoxy)phosphazene-containing particle(s) according to
the invention, such particles can be formed using any way known or to be
developed in the art. Two exemplary preferred methods of accomplishing
this include wherein (i) the non-solvent residing in the vessel in the
method embodiment described above is cooled to close to its freezing
point or to its freezing point prior to the addition of the polymer
solution such that the polymer droplets freeze upon contact with the
pre-cooled non-solvent; or (ii) the polymer droplets are frozen by
contacting them with a liquefied gas such as nitrogen, which is placed
over a bed of pre-frozen non-solvent (see, FIG. 2). In method (ii), after
the nitrogen evaporates, the non-solvent slowly thaws and the
microspheres in their frozen state will sink into the liquid, cold
non-solvent where the extraction process (removal of the polymer solvent)
will be carried out.

[0059]By modifying this general process, one may prepare particles that
are hollow or substantially hollow or porous. For example, if the removal
of the solvent from the bead is carried out quickly, e.g., by applying a
vacuum during the final stage of incubation, porous beads will result.

[0060]The particles of the invention can be prepared in any size desired,
"Microspheres" may be obtained by nebulizing the polymer solution into a
polymer aerosol using either pneumatic or ultrasonic nozzles, such as,
for example a Sonotek 8700-60 ms or a Lechler US50 ultrasonic nozzle,
each available from Sono[.tek] Corporation, Milton, N.Y., U.S.A. and
Lechler GmbH, Metzingen, Germany. Larger particles may be obtained by
dispensing the droplets into the non-solvent solution using a syringe or
other drop-forming device. Moreover, as will be known to a person of
skill in the art, the size of the particle may also be altered or
modified by an increase or decrease of the initial concentration of the
polymer in the polymer solution, as a higher concentration will lead to
an increased sphere diameter.

[0061]In an alternative embodiment of the particles described herein, the
particles can include a standard and/or a preferred core based on an
acrylic polymer or copolymer with a shell of
poly[bis(trifluoroethoxy)phosphazene. Such particles can provide a
preferred spherical shape and improved specific gravity for use in a
suspension of injection or delivery medium. The acrylic polymer based
polymers with poly[bis(trifluoroethoxy)phosphazene shell described herein
provide a substantially spherical shape, mechanical flexibility and
compressibility, improved specific gravity properties. The core polymers
may be formed using any acceptable technique known in the art, such as
that described in B. Thanoo et al., "Preparation of Hydrogel Beads from
Crosslinked Poly(Methyl Methacrylate) Microspheres by Alkaline
Hydrolysis," J. Appl. P. Sci., Vol. 38, 1153-1161 (1990), incorporated
herein by reference with respect thereto. Such acrylic-based polymers are
preferably formed by polymerizing unhydrolyzed precursors, including,
without limitation, methyl acrylate (MA), methyl methacrylate (MMA),
ethylmethacrylate (EMA), hexamethyl (HMMA) or hydroxyethyl methacrylate
(HEMA), and derivatives, variants or copolymers of such acrylic acid
derivatives. Most preferred is MMA. The polymer should be present in the
core in a hydrated or partially hydrated (hydrogel) form. Such polymers
are preferably cross-linked in order to provide suitable hydrogel
properties and structure, such as enhanced non-biodegradability, and to
help retain the mechanical stability of the polymer structure by
resisting dissolution by water.

[0062]Preferably, the core prepolymers are formed by dispersion
polymerization that may be of the suspension or emulsion polymerization
type. Emulsion polymerization results in substantially spherical
particles of about 10 nm to about 10 microns. Suspension polymerization
results in similar particles but of larger sizes of about 50 to about
1200 microns.

[0063]Suspension polymerization may be initiated with a thermal initiator,
which may be solubilized in the aqueous or, more preferably, monomer
phase. Suitable initiators for use in the monomer phase composition
include benzoyl peroxide, lauroyl peroxide or other similar
peroxide-based initiators known or to be developed in the art, with the
most preferred initiator being lauroyl peroxide. The initiator is
preferably present in an amount of about 0.1 to about 5 percent by weight
based on the weight of the monomer, more preferably about 0.3 to about 1
percent by weight based on the weight of the monomer. As noted above, a
cross-linking co-monomer is preferred for use in forming the hydrated
polymer. Suitable cross-linking co-monomers for use with the
acrylic-based principle monomer(s) used in preparing a polymerized
particle core, include various glycol-based materials such as ethylene
glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA)
or most preferably, triethylene glycol dimethacrylate (TEGMDA). A chain
transfer agent may also be provided if desired. My suitable MA
polymerization chain transfer agent may be used. In the preferred
embodiment herein, dodecylmercaptane may be used as a chain transfer
agent in amounts acceptable for the particular polymerization reaction.

[0064]The aqueous phase composition preferably includes a
surfactant/dispersant as well as a complexing agent, and an optional
buffer is necessary. Surfactants/dispersants should be compatible with
the monomers used herein, including Cyanamer® 370M, polyacrylic acid
and partially hydrolyzed polyvinyl alcohol surfactants such as 4/88,
26/88, 40/88. A dispersant should be present in an amount of about 0.1 to
about 5 percent by weight based on the amount of water in the dispersion,
more preferably about 0.2 to about 1 percent by weight based on the
amount of water in the dispersion. An optional buffer solution may be
used if needed to maintain adequate pH. A preferred buffer solution
includes sodium phosphates (Na2HPO4/NaH2PO4). A
suitable complexing agent is ethylene diamine tetraacetic acid (EDTA),
which may be added to the aqueous phase in a concentration of from about
10 to about 40 ppm EDTA, and more preferably about 20 to about 30 ppm. It
is preferred that in the aqueous phase composition, the monomer to water
ratio is about 1:4 to about 1:6.

[0065]The polymerization should take place at about ambient conditions,
preferably from about 60° C. to about 80° C. with a time to
gelation of about one to two hours. Stirring at rates of 100 to 500 rpm
is preferred for particle formation, with lower rates applying to larger
sized particles and higher rates applying to smaller sized particles.

[0066]Once PMMA particles, such as microparticles, are formed, they are
preferably subjected to hydrolysis conditions typical of those in the
art, including use of about 1-10 molar excess of potassium hydroxide per
mol of PMMA. Such potassium hydroxide is provided in a concentration of
about 1-15% potassium hydroxide in ethylene glycol. The solution is then
heated preferably at temperatures of about 150-185° C. for several
hours. Alternatively, to minimize reactant amounts and cost, it is
preferred that lesser amounts of potassium hydroxide be used which are
less than about 5 molar excess of potassium hydroxide per mole of PMMA,
more preferably about 3 molar excess or less. For such hydrolytic
reactions, a concentration of about 10-15% potassium hydroxide in
ethylene glycol is also preferably used, and more preferably about 14% to
about 15%. It will be understood by one skilled in the art, that heating
conditions at higher temperatures may be used to decrease overall
reaction times. Reaction times may be varied depending on the overall
diameter of the resultant particles. For example, the following
conditions are able to provide particles having about 35% compressibility
and desired stability: for diameters of about 200-300 μm, the solution
should be heated for about 7.5 to about 8.5 hours; for diameters of about
300-355 μm, about 9.5 to about 10.5 hours; for diameters of about
355-400 μm, about 11.5 to about 12.5 hours; and for about 400-455
μm, about 13.5 to about 14.5 hours, etc. The particle size can be
adjusted using variations in the polymerization process, for example, by
varying the stirring speed and the ratio of the monomer to the aqueous
phase. Further, smaller sizes can be achieved by increasing
surfactant/dispersant ratio.

[0067]Following hydrolysis, particles are separated from the reaction
mixture and their pH may be adjusted to any range as suited for further
processing steps or intended uses. The pH of the particle core may be
adjusted in from about 1.0 to about 9.4, preferably about 7.4 if intended
for a physiological application. Since size, swelling ratio and
elasticity of the hydrogel core material are dependent on pH value, the
lower pH values may be used to have beneficial effects during drying to
prevent particle agglomeration and/or structural damage. Particles are
preferably sieved into different size fractions according to intended
use. Drying of particles preferably occurs using any standard drying
process, including use of an oven at a temperature of about
40°-80° C. for several hours up to about a day.

[0068]To provide desired surface properties to the hydrophilic hydrogel
particles, in order to provide adhesion for receiving a
poly[bis(trifluoroethoxy)phosphazene coating, the surface of the hydrogel
may be subjected to treatment with any suitable ionic or non-ionic
surfactant, such as tetraalkylammonium salts, polyalcohols and similar
materials. A more permanent change in adhesion properties is brought
about by rendering the surface of the particles hydrophobic by reaction
of its polymethacrylic acid groups with a suitable reactant. Suitable
reactants include, but are not limited to, hydrophobic alcohols, amides
and carboxylic acid derivatives, more preferably they include halogenated
alcohols such as trifluoroethanol. Such surface treatment also prevents
delamination of the coating from the core once the coating is applied.
Preferred surface treatments may include, without limitation, an initial
treatment with thionyl chloride followed by reaction with
trifluoroethanol. Alternatively, the surface may be treated by suspending
the particles in a mixture of sulfuric acid and a hydrophobic alcohol,
such as trifluoroethanol. Such treatments are preferred if the particles
are to be coated in that they minimize any delamination of a coating.

[0069]Alternatively, and most preferably, the PMA core particles may be
coated with a surface layer of and/or infused with barium sulfate. The
barium sulfate is radiopaque and aids in visualization of the finished
particles when in use. It also provides enhanced fluidization properties
to the particles such that it reduces agglomeration especially during
drying and allows for fluid bed coating of the PMA particles with an
outer coating of poly[bis(trifluoroethoxy)phosphazene], thereby providing
improved adhesion between a poly[bis(trifluoroethoxy)phosphazene] outer
core and a polymeric acrylate core particles. By allowing fluidization
even when the core particles are swollen, barium sulfate also improves
the overall coating and adhesion properties. By enabling the coating of
the core particles even in a swollen state with
poly[bis(trifluoroethoxy)phosphazene, barium sulfate also reduces the
potential tendency of the poly[bis(trifluoroethoxy)phosphazene shells to
crack or rupture in comparison with coating the particles in a dry state
and then later exposing the particles to a suspension in which the core
particles swell and exert force on the shell of
poly[bis(trifluoroethoxy)phosphazene. A coating of barium sulfate on the
core particles is preferably applied by adhesion of the barium sulfate in
the form of an opaque coating on the hydrogel surface of the PMA beads.
Barium sulfate can further assist in reducing electrostatic effects that
limit particle size. By allowing for absorption of additional humidity,
the barium sulfate tends to counteract the electrostatic effects.

[0070]Barium sulfate crystals adhering only loosely to the PMA particles
may be covalently crosslinked or chemically grafted to the particle
surface by spraycoating a sufficient amount of an aminosilane adhesion
promoter onto the PMA particle. This will help to effectively reduce
barium sulfate particulate matter in solution after hydration of the
particles. Exemplary particles include 3-aminopropyl-trimethoxysilane and
similar silane-based adhesion promoters, such as, for example,
N-methyl-aza-2,2,4-trimethylsilacyclopentane,
2,2-dimethoxy-1,6-diaza-2-silacyclooctane,
(3-trimethoxysilylpropyl)diethylene triamine,
N-(3-(trimethoxysilyl)propyl)methanediamine,
N1,N2-bis(3-(trimethoxysilyl)propyl)ethane-1,2-diamine,
1,3,5-tris(3-(trimethoxysilyl)propyl)-1,3,5-triazinane-2-4-6-trione, and
similar silane-based adhesion promoters.

[0071]A further alternative for improving visualization of microparticles
made as noted herein include the absorption of a water soluble organic
dye inside the hydrogel core particles. Exemplary dyes are preferably
those FDA dyes approved for human use and which are known or to be
developed for safe, non-toxic use in the body and which are capable of
providing acceptable contrast. Organic dyes may include dyes such as D&C
Violet no. 2 and others preferably approved for medical device uses, such
as for contact lenses and resorbable sutures. Whereas barium sulfate
operates as an inorganic filler and finely dispersed pigment that makes
the particles visible by light diffraction due to small crystal size, the
dyes when impregnated in the particles absorb the complementary part of
the visible color spectrum.

[0072]Particles, including microparticles made in accordance with the
foregoing process for forming a core hydrogel polymer are then coated
with poly[bis(trifluoroethoxy)phosphazene and/or its derivatives. Any
suitable coating process may be used, including solvent fluidized bed
and/or spraying techniques. However, preferred results may be achieved
using fluidized bed techniques in which the particles pass through an air
stream and are coated through spraying while they spin within the air
stream. The poly[bis(trifluoroethoxy)phosphazene or derivative polymer is
provided in dilute solution for spraying to avoid clogging of the nozzle.

[0073]Exemplary solvents for use in such solutions include ethyl acetate,
acetone, hexafluorbenzene, methyl ethyl ketone and similar solvents and
mixtures and combinations thereof, most preferred is ethyl acetate alone
or in combination with isoamyl acetate. Typical preferred concentrations
include about 0.01 to about 0.3 weight percent
poly[bis(trifluoroethoxy)phosphazene or its
poly[bis(trifluoroethoxy)phosphazene derivative in solution, more
preferably about 0.02 to 0.2 weight percent
poly[bis(trifluoroethoxy)phosphazene, and most preferably about 0.075 to
about 0.2 weight percent. It should be understood based on this
disclosure that the type of hydrogel core can be varied as can the
technique for coating a particle, however it is preferred that a core
which is useful in the treatment techniques and applications described
herein is formed and subsequently coated with
poly[bis(trifluoroethoxy)phosphazene] and/or its derivatives as described
herein.

[0074]For use in embodiments of the present invention, particle density is
preferably taken into consideration to ensure beneficial properties for
particle delivery. Possible clogging of a catheter-based delivery system
may occur if using a density-mismatched delivery medium. In addition, it
is desirable to include a certain minimum amount of contrast agent in the
delivery medium to achieve sufficient levels of fluoroscopic contrast
during surgery. Currently, the polymethacrylate hydrogel density is
between 1.05 g/cm3 and 1.10 g/cm3 depending on the equilibrium
water content. The most common iodinated nonionic contrast agent media
with 300 mg iodine per ml have densities of 1.32-1.34 g/cm3. As used
herein, "buoyancy" refers to the ability of the particles to be
substantially free floating in solution that occurs when the density of
the particle is substantially the same as the medium in which it is
suspended. Coated particles formed in accordance with the present
invention as described herein can reach buoyancy when there is
approximately 30% contrast agent in the delivery medium, however, such
levels can be adjusted for such preferred use according to techniques
described herein.

[0075]One method for increasing the density of the particles is by use of
heavy water or deuterium oxide (D2O). When heavy water is used to
swell the particles, D2O displaces H2O, thereby increasing the
weight of the particles for better dispersion and buoyancy levels.
Typically this leads to the ability to add higher amounts of contrast
agent of at least about 5% using such a technique. However, some
equilibrating effect can occur over time when the particles are contacted
with an aqueous solution of contrasting agent. Thus, it is preferred that
when using D2O for this purpose, either that suspension times are
kept to a minimum or, more preferably, that the contrast agent be
provided in a solution which also uses D2O.

[0076]Alternatively, particles of pH 1 can be neutralized with cesium
hydroxide and/or the final neutralized particles can be equilibrated with
cesium chloride. Such compounds diffuse cesium into the particles, such
that either the cesium salt of polymethacrylic acid is formed or
polymethacrylic acid is diffused and thereby enriched with cesium
chloride.

[0077]The cesium increases the density of the particles, thereby
increasing the ability to add higher amounts of contrast agent. Typical
buoyancy levels can be adjusted using the cesium technique such that
about 45 to about 50% contrast agent may be added to the delivery medium
as is desired. Cesium salts are non-toxic and render the particles
visible using fluoroscopy. Cesium's atomic weight of 132.9 g/mol is
slightly higher than that of iodine providing beneficial effects
including increase in overall density and enhancement of X-ray contrast
visibility even without a contrast agent. For certain cancer treatments
where a radioactive isotope of cesium is desired, such active agent can
be used as an alternative cesium source rendering the particles buoyant
in an injectable solution as well as able to be used as an active
treatment source.

[0078]The above-noted techniques for improving density of particles, such
as microparticles for applications where density and/or buoyancy in
solution are applicable properties may be applied in to the preferred
particles described herein and/or may be applied for other similar
particles. It should be understood that the disclosure is not limited to
cesium and/or D2O treatment of the preferred particles herein and
that such techniques may have broader implications in other particles
such as other acrylic-based hydrogels and other polymeric particles.

[0079]As noted above, barium sulfate may be used between the core
particles and the preferred poly[bis(trifluoroethoxy)phosphazene coating
or introduced into the interior of the core particles using any technique
known or to be developed in the art. Also, organic dyes may similarly be
included in the particle core. These materials, particularly the barium
sulfate, also contribute to an increase in density as well as providing
radiopacity. In addition to a general density increase as provided by the
above-noted D2O or cesium compounds, the barium sulfate allows this
benefit even upon substantial and/or full hydration, allowing particles
in suspension to remain isotonic. Thus, a barium sulfate powder coating
can provide an inert precipitate having no effect on physiological
osmolarity.

[0080]It should be understood, based on this disclosure, that the various
buoyancy additives noted above can be used independently or in
combination to provide the most beneficial effects for a given core
particle and coating combination.

[0081]The invention also includes methods of delivering an active agent to
a localized bone defect within the body of a mammal. The method includes
contacting the localized area with at least one of the particles of the
invention as described above, such that an effective amount of the active
agent is released locally to the area. As used herein, "contact" or
"contacting" the localized area with at least one particle is intended to
include situating at least one particle and an active agent in
sufficiently close proximity to the intended area such that the desired
effect is achieved. Diseases or pathologies that may be treated by this
method include any wherein the localized or topical application of the
active agent achieves some benefit in contrast to the systemic absorption
of the drug. Suitable active agents include NSAIDS, steroids, hormones,
nucleic acids, antibiotics, antiseptics, osteogenesis-enhancing agents,
analgesics, anesthetics, or biological agents to promote regrowth of bone
within the defect.

[0082]According to the present invention, one or more active agents may be
delivered by using the hydrogel core of the microspheres as described
herein as a delivery vehicle. Alternately or in conjunction with use of
the hydrogel core as a drug delivery vehicle for active agents, active
agents may also be delivered with microspheres of the present invention
into defects in bone as a component of the injection or delivery medium,
or instilled into such bone defects following delivery of the
microspheres of the present invention.

[0083]As used herein, the terms "antiseptic agents" and "antiseptics,"
which terms may be used interchangeably herein, are substances which may
be used to reduce microbial levels and are biologically compatible enough
to be applied to a particular anatomic surface without causing
substantial irritation, inflammation, dysfunctional or other undesired
reactions on or within the anatomic surface or adjacent tissues or
organs. Antiseptic agents used in compositions according to the present
invention may be microbicidal (bacteriocidal, fungicidal, and/or
viricidal) in their actions, and are intended to provide a reduction in
the ambient flora in the anatomic surface onto which they are
administered.

[0084]As further used herein, the terms "antibiotic" and "antibiotics" are
substances that are capable of destroying or weakening certain
microorganisms, especially bacteria or fungi that may cause infections or
infectious diseases. Antibiotics may be produced by or synthesized from
other microorganisms, or they may be entirely synthetic compounds.
Antibiotics may inhibit pathogenesis by interfering with essential
intracellular processes, including the synthesis of bacterial proteins.

[0086]As further used herein, the terms "anesthetic agents" and
"anesthetics," which terms may be used interchangeably herein, are
substances which may be used to induce anesthesia, or reversibly depress
neuronal function, producing total or partial loss of pain sensation when
administered to an anatomic surface or tissue. Anestheics which may be
used in the present invention include, but are not limited to, lidocaine,
prilocaine, bupivacaine, mepivacaine and related local anesthetic
compounds having various substituents on the ring system or amine
nitrogen; the aminoalkyl benzoate compounds, such as procaine,
chloroprocaine, propoxycaine, hexylcaine, tetracaine, cyclomethycaine,
benoxinate, butacaine, proparacaine, and related local anesthetic
compounds; cocaine and related local anesthetic compounds; amino
carbonate compounds such as diperodon and related local anesthetic
compounds; N-phenylamidine compounds such as phenacaine and related
anesthetic compounds; N-aminoalkyl amid compounds such as dibucaine and
related local anesthetic compounds; aminoketone compounds such as
falicaine, dyclonine and related local anesthetic compounds; and amino
ether compounds such as pramoxine, dimethisoquien, and related local
anesthetic compounds., and derivatives, metabolites, and/or combinations
thereof.

[0087]As further used herein, the terms "analgesic" and "analgesics" are
pharmaceutical compounds capable of reducing or eliminating pain when
administered to a mammalian patient. Analgesics which may be used in the
present invention include, but are not limited to, morphine, fentanyl,
sufentanil, remifentanil, and other opioids, tramadol, other non-opioid
analgesics, and derivatives, metabolites, and/or combinations thereof.

[0088]Compositions of the present invention may comprise povidone-iodine
as an antiseptic agent in a effective concentration of about 0.5%, about
1%, about 2.5%, about 5%, about 7.5%, about 10%, about 12.5%, about 15%,
about 17.5%, and about 20%, where "about" means±1.25%.

[0090]Alternatively, the microspheres of the present invention may be
mixed with an inorganic filler. The inorganic filler may be selected from
alphatricalcium phosphate, beta-tricalcium phosphate, calcium carbonate,
barium carbonate, calcium sulfate, barium sulfate, hydroxyapatite, and
mixtures thereof. In certain embodiments the inorganic filler comprises a
polymorph of calcium phosphate. Such organic fillers according to the
present invention may be incorporated in the hydrogel core of the
microspheres, or delivered as a component of the injection/delivery
medium used with the microspheres. In a preferred embodiment of the
present invention, the inorganic filler is hydroxyapatite.

[0091]Embodiments of the present invention may further include bone
adhesives in the injection or delivery medium, or such a bone adhesive
may be instilled after delivery of the microspheres to the bone defect
being treated. The term "bone adhesive" is used collectively herein to
include bone glues and bone cements. The bone glues which can be used in
the practice of the present invention include conventional biocompatible
bone glues including 2-octyl cyanoacrylate and the like and equivalent
thereof. The bone cements which can be used in the practice of the
present invention include conventional biocompatible bone cements such as
polymethylmethacrylate and the like. The bone glues and bone cements in
the present invention may be absorbable or nonabsorbable.

[0092]If the particle formulated for delivery of an active agent to a
localized area is about 1 to about 1,000 μm in diameter, the drug
loaded microspheres can be applied to localized areas within the
mammalian body using syringes and/or catheters as a delivery device,
without causing inadvertent occlusions. As will be understood to a person
of skill in the art, injection mediums include any pharmaceutically
acceptable mediums that are known or to be developed in the art, such as,
e.g., saline, PBS or any other suitable physiological medium. In
accordance with a further embodiment described herein, the invention
includes an injectible dispersion including particles and a contrasting
agent which particles are substantially dispersed in the solution. In a
preferred embodiment, the particles are also detectible through
fluoroscopy.

[0093]Moreover, it is envisioned that the active agent can be selected so
as to complement the action of the particles in a synergistic fashion,
especially if the particles are being used in an infectious or
metaplastic application. For example, if the bone defect being treated is
due to an osseous tumor, one may wish to load the particles used with a
cytostatic drug, antiangiogenic agents, or an antimitotic drug.
Alternately, if the bone defect being treated is due to an osteomyelitis,
one may wish to load the particles used with one or more appropriate
antibiotic agents.

[0094]In further alternative embodiments of the present invention, a
coating of poly[bis(trifluoroethoxy)phosphazene may be applied to
bioceramic microspheres, rather than to the hydrogel core previously
described. In such embodiments, the bioceramic microspheres may be any
biocompatible ceramic material including, but not limited to,
hydroxyapatite ceramics, β-tricalcium phosphate ceramics, biphasic
calcium phosphate ceramics, macroporous ceramics, and calcium phosphate
hydraulic cements.

[0095]In other aspects, certain calcium phosphate cement compositions may
be employed in this invention. For example, calcium tertiary phosphate,
calcium secondary phosphate, or a combination thereof may be a useful
cement composition. In one aspect, the hydraulic calcium phosphate cement
composition disclosed in U.S. Pat. No. 5,152,836, which contains as main
ingredients powders of calcium tertiary phosphate and calcium secondary
phosphate with a molar ratio of Ca/P of 1.400 to 1.498 may be useful.
This hydraulic calcium phosphate cement composition comprising as main
ingredients powders of calcium tertiary phosphate and calcium secondary
phosphate with a molar ratio of Ca/P of 1.400 to 1.498, in which the
calcium tertiary phosphate containing α-type calcium tertiary
phosphate and β-type calcium tertiary phosphate. In another aspect,
the α-type and β-type calcium tertiary phosphates may be
combined in a ratio of, for example, from about 97:3 to 50:50 by weight,
to provide the cement composition. Other hydraulic calcium phosphate
cement compositions such as those disclosed in Biomaterials, vol. 25
(7-8), 1439-1451 (2004) may also be used in this invention. Thus,
dicalcium phosphate dihydrate (DCPD) hydraulic cement and apatite
hydraulic cement can be used as well. In a further aspect, the core can
comprise a hydrogel, a ceramic, a metal, an alloy, or a metal compound,
and the like. For example, metals or metal alloys that include scandium,
yttrium, lanthanum, titanium, zirconium, hafnium, vanadium, niobium,
tantalum, chromium, molybdenum, tungsten, manganese, technetium, rhenium,
iron, ruthenium, osmium, cobalt, rhodium, iridium, nickel, palladium,
platinum, copper, silver, gold, aluminum, gallium, silicon, germanium,
rare earth metals, lanthanides, actinides, transition metals, and the
like, including combinations thereof, alloys thereof may be employed.

[0096]The present invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be clearly
understood that resort can be had to various other aspects, embodiments,
modifications, and equivalents thereof which, after reading the
description herein, can suggest themselves to one of ordinary skill in
the art without departing from the spirit of the present invention or the
scope of the appended claims.

[0097]Further, it is to be understood that this invention is not limited
to specific materials, agents, ceramics, cements, polyphosphazenes, or
other compounds used and disclosed in the invention described herein,
including in the following examples, as each of these can vary. It is
also to be understood that the terminology used herein is for the purpose
of describing particular aspects or embodiments and is not intended to be
limiting. Should the usage or terminology used in any reference that is
incorporated by reference conflict with the usage or terminology used in
this disclosure, the usage and terminology of this disclosure controls.

[0098]Unless indicated otherwise, temperature is reported in degrees
Centigrade and pressure is at or near atmospheric. An example of the
preparation of a polyphosphazene of this invention is provided with the
synthesis of poly[bis(trifluoroethoxy)phosphazene] (PzF) polymer, which
may be prepared according to U.S. Patent Application Publication No.
2003/0157142, the entirety of which is hereby incorporated by reference.

[0099]Also unless indicated otherwise, when a range of any type is
disclosed or claimed, for example a range of molecular weights, layer
thicknesses, concentrations, temperatures, and the like, it is intended
to disclose or claim individually each possible number that such a range
could reasonably encompass, including any sub-ranges encompassed therein.
For example, when the Applicants disclose or claim a chemical moiety
having a certain number of atoms, for example carbon atoms, Applicants'
intent is to disclose or claim individually every possible number that
such a range could encompass, consistent with the disclosure herein.
Thus, by the disclosure that an alkyl substituent or group can have from
1 to 20 carbon atoms, Applicants intent is to recite that the alkyl group
have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 carbon atoms. In another example, by the disclosure that
microspheres have a diameter of approximately 500 to 600 μm,
Applicants include within this disclosure the recitation that the
microspheres have a diameter of approximately 500 μm, approximately
510 μm, approximately 520 μm, approximately 530 μm,
approximately 540 μm, approximately 550 μm, approximately 560
μm, approximately 570 μm, approximately 580 μm, approximately
590 μm, and/or approximately 600 μm, including any range or
sub-range encompassed therein. Accordingly, Applicants reserve the right
to proviso out or exclude any individual members of such a group,
including any sub-ranges or combinations of sub-ranges within the group,
that can be claimed according to a range or in any similar manner, if for
any reason Applicants choose to claim less than the full measure of the
disclosure, for example, to account for a reference that Applicants are
unaware of at the time of the filing of the application.

EXAMPLE 1

[0100]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] polymer of a molecular weight
3×106 g/mol in the polymer solvent ethyl acetate to obtain a
2% (wt/v) polymer solution. Four milliliters of this polymer solution was
manually dripped into liquid nitrogen using a 5 ml syringe. This
dispersion was dispensed onto a frozen layer of 150 milliliters of
pentane. (See FIG. 2.) The cryoextraction was allowed to proceed for
three days. Subsequently, polymeric particles were retrieved from the
reaction vessel, and were air dried at 21° C.

EXAMPLE 2

[0101]Microspheres having a diameter of approximately 350 to 450 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] polymer of a molecular weight
3×106 g/mol in ethyl acetate to obtain a 1% (wt/v) polymer
solution. Four milliliters of this polymer solution was manually dripped
into liquid nitrogen using a 5 ml syringe. This dispersion was dispensed
onto a frozen layer of 150 milliliters of pentane. (See FIG. 2.) The
cryoextraction was allowed to proceed for three days. Subsequently,
polymeric particles were retrieved from the reaction vessel and were air
dried at 21° C.

EXAMPLE 3

[0102]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] of a molecular weight
12×106 g/mol in methylisobutylketone to obtain a 2% (wt/v)
polymer solution. Four milliliters of this polymer solution was manually
dripped into liquid nitrogen using a 5 ml syringe. This dispersion was
dispensed onto a frozen layer of 150 milliliters of a 1:9 (v/v)
ethanol/pentane mixture (See FIG. 2.). The cryoextraction was allowed to
proceed for three days. Subsequently, polymeric particles were retrieved
from the reaction vessel, and dried under reduced pressure at 21°
C.

EXAMPLE 4

[0103]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] of a molecular weight
9×106 g/mol in isoamylketone to obtain a 2% (wt/v) polymer
solution. Four milliliters of this polymer solution was manually dripped
into liquid nitrogen using a 5 ml syringe. This dispersion was dispensed
onto a frozen layer of 150 milliliters of pentane. (See FIG. 2.) The
cryoextraction was allowed to proceed for three days. Subsequently,
polymeric polymers were retrieved from the reaction vessel and dried
under reduced pressure at 21° C.

EXAMPLE 5

[0104]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] of a molecular weight
16×106 g/mol in cyclohexanone to obtain a 2% (wt/v) polymer
solution. Four milliliters of this polymer solution was manually dropped
into liquid nitrogen using a 5 ml syringe. This dispersion was dispensed
onto a frozen layer of 150 milliliters of a 1:1 (v/v) ethanol/diethyl
ether mixture. (See FIG. 2.) The cryoextraction was allowed to proceed
for three days. Subsequently, polymeric particles were retrieved from the
reaction vessel and dried under reduced pressure at 21° C.

EXAMPLE 6

[0105]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] polymer of a molecular weight
3×106 g/mol in ethyl acetate to obtain a 2% (wt/v) polymer
solution. Four milliliters of this polymer solution was manually dripped
into liquid nitrogen using a 5 ml syringe. This dispersion was dispensed
onto a frozen layer of 150 milliliters of hexane. (See FIG. 2.) The
cryoextraction was allowed to proceed for three days. Subsequently,
polymeric particles were retrieved from the reaction vessel and air dried
at 21° C.

EXAMPLE 7

[0106]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] of a molecular weight
3×106 g/mol in ethyl acetate to obtain a 2% (wt/v) polymer
solution. Four milliliters of this polymer solution was manually dripped
into liquid nitrogen using a 5 ml syringe. This dispersion was dispensed
onto a frozen layer of 150 milliliters of ethanol. (See FIG. 2.) The
cryoextraction was allowed to proceed for three days. Subsequently,
polymeric particles were retrieved from the reaction vessel and air dried
at 21° C. The particles were noticeably gel-like and after drying
were ellipsoid in shape.

EXAMPLE 8

[0107]Microspheres having a diameter of approximately 500 to 600 μm
were prepared. First, a polymer solution was prepared by dissolving
poly[bis(trifluoroethoxy)phosphazene] of a molecular weight
3×106 g/mol in ethyl acetate to obtain a 2% (wt/v) polymer
solution. Four milliliters of this polymer solution was manually dripped
into liquid nitrogen using a 5 ml syringe. This dispersion was dispensed
onto a frozen layer of 150 milliliters of diethylether. (See FIG. 2.) The
cryoextraction was allowed to proceed for three days. Subsequently,
polymeric particles were retrieved from the reaction vessel and air dried
at 21° C. The resultant particles were, after drying, compact and
uniformly spherical.

EXAMPLE 9

[0108]A two liter cryovessel as shown in FIG. 6 was filled with 100
milliliters of diethyl ether as a non-solvent. Liquid nitrogen was slowly
added until the non-solvent froze. The vessel was then filled with
additional liquid nitrogen, until the amount of liquid nitrogen rose
approximately 5 to 10 cm when measured vertically above the non-solvent
layer. The vessel was closed with an insulated lid, and a syringe needle
connected via Teflon tubing to a syringe pump was inserted through a
small opening in the lid.

[0109]The syringe pump as shown in FIG. 7, was used to dispense between 5
to 15 milliliters of the 5 to 40 mg/ml polymer solution in ethyl acetate,
slowly into the cryovessel. The rate of the pump was adjusted to
approximately 10 milliliters dispensing volume per hour. A Teflon®
cylinder with one inlet and one to eight outlets is used to distribute
the dispensed volumes into several vessels in parallel. (It is preferable
that the ratio of solvent to non-solvent volume stays below 10% (v/v).
Otherwise the particles may adhere to one another.) After the polymer
solution was completely dispensed into the vessel, another 100
milliliters of non-solvent was slowly poured on top of the liquid
nitrogen.

[0110]In carrying out this process, it is noted that it is preferable that
the needle tips used for dispensing are small, such as the G33 size.
Additionally, the dropping distance should be more than 5 cm, so that the
droplets aided by gravity immediately sink into the liquid nitrogen upon
hitting the surface.

[0111]The liquid nitrogen in the vessel was slowly allowed to evaporate,
taking approximately one day. The non-solvent slowly began to melt, and
the polymer solution droplets, still frozen, sank into the cold
non-solvent. After another day of incubation, the now gelled polymer
beads (articles) were retrieved from the vessel by simple filtration.
They were allowed to dry at room temperature for approximately 30 minutes
and then were ready for use in any of the applications described herein.

EXAMPLE 10

[0112]The microspheres prepared by the process of Example 1 were examined
for shape and surface morphology by optical microscope, scanning electron
microscope (SEM) and atomic force microscopy. The results of these
analyses are shown in FIGS. 3A and 3B). FIG. 3A shows the microspheres as
they appear using an optical microscope at 4× magnification. FIG.
3B shows a microsphere as it appears using a scanning electron microscope
at 100× magnification.

[0113]It can be seen that surface morphology of the unloaded spheres is
typical for semi-crystalline polymers above glass transition temperature.
Amorphous as well crystalline regions are prevalent throughout the sample
surface. The surface is microporous in nature, with pore sizes ranging
from nanometers to few micrometers in diameter.

[0114]Particles loaded with bovine insulin were also analyzed using
scanning electron microscopy (100× magnification). The result of
these analyses can be seen in FIGS. 4A and FIG. 4B).

EXAMPLE 11

[0115]Several polymerizations were carried out using varying combinations
of PMMA and three different crosslinking monomers (EDGMA, DEGDMA and
TEGDMA), different radical initiators (benzoyl peroxide (BPO) and lauroyl
peroxide (LPO), EDTA as a complexing agent and varying dispersants
(Cyanamer 370M, polyacrylic acid (PAA) and varying types of polyvinyl
alcohol (PVA) to achieve the preferred core particles. In some
polymerizations, sodium phosphate buffer solution
(Na2HPO4/NaH2PO4) was used. It was observed that some
of the reaction procedures went unsuccessful due to the type of
dispersant and concentration chosen. Failure of the dispersant was
demonstrated in the form of early onset of an exothermic reaction,
coalescing aqueous and organic phases and premature onset of the
vitrification phase. Only the successful examples are shown. The
successful runs are shown below in Table 1, which includes the
components, concentrations and reaction conditions for such samples
(1-6).

[0116]Hydrogel microparticles formed in accordance with the procedures
described herein were evaluated for buoyancy and suspension properties
for use in injection applications. The microparticles included a sample
using unmodified polymethacrylic acid potassium salt hydrogel particles
(Sample A); a sample using trifluoroethyl esterified polymethacrylic acid
potassium salt hydrogels (Sample B); and a sample using the same hydrogel
as Sample B, but wherein the particles were coated with
poly[bis(trifluoroethoxy)phosphazene] (Sample C). An isotonic phosphate
buffered saline solution of pH 7.4 having 0.05 volume % Tween® 20 was
prepared by dissolving 5 phosphate buffered saline tablets (Fluka®)
in 999.5 ml of milliQ ultrapure water. 0.5 ml of Tween 20® surfactant
was added to the solution. Solutions having between 20 and 50 percent by
volume of Imeron300® contrast agent in the isotonic buffered saline
solution were then prepared for evaluation.

[0117]The contrast agent solutions which were prepared were then placed in
4 ml vials in aliquots of 2 ml each. To the vials, 50-80 mg of the
hydrated hydrogel Samples A-C were added. Each Sample was first hydrated
by adding to 100 mg of dry hydrogel microparticles either 900 mg of
isotonic phosphate buffered saline solution or D2O to obtain 1 ml
swollen hydrogel. Buoyancy properties were measured immediately and every
10 minutes thereafter until buoyancy equilibrium was achieved and/or
surpassed.

[0118]All of the particles reached equilibrium density in the contrast
agent solution having 30-40% contrasting agent within 5 min. Particles
which were swollen with D2O were heavier within the first 10
minutes, but the D2O did diffuse out of the particles over time
within 15-20 min. of immersion. If additional water which could displace
the D2O were not added, microparticles hydrated with D2O would
be able to increase the contrast agent percentage achievable with
adequate buoyancy by as much as 5%. Particles began to float to the top
over time when the contrast agent was added in percentages of 40%-50%.

[0119]The equilibrium buoyancy (matching densities) was achieved for
Sample C in 31±1 volume percent of contrast agent in solution. With
regard to Samples A and B, swelling behavior and subsequent density are
typically dependent on crosslinking content, pH, ionic strength and
valence of cations used. However, it was assumed herein that the swelling
does not influence buoyancy due to the sponge-like nature of the
polymethacrylic acid hydrogel material. After such material was coated
with the poly[bis(trifluoroethoxy)phosphazene] as in Sample C, a time lag
of swelling was observed and buoyancy equilibrium was slower to achieve.

EXAMPLE 13

[0120]In order to take account of the time lag and to achieve a more
preferred density, as well as to enhance the fluoroscopic visibility of
the particles, cesium treatment was then effected for the types of
microparticles used in Samples B and C of Example 12.

[0121]100 mg of Sample C and of Sample B were hydrated each for 10 min. in
a 30 weight percent solution of sodium chloride. The supernatant liquid
was decanted after equilibrium and the microparticles were washed
thoroughly with deionized water. They were then equilibrated for another
10 min., decanted and suspended in 3 ml of surfactant-free isotonic
phosphate buffer solution at a pH 7.4. The effect on buoyancy was then
evaluated using contrast agent solutions varying from 20 to 50% by volume
of Imeron® 300. In this Example, 0.1 g of the microparticles of
Samples B and C were used. 3.5 ml of Imeron 300 contrast agent were
provided to the initial buffer solution which included 4.0 ml isotonic
phosphate buffer/Tween® 20 solution.

[0122]The equilibration procedure using cesium chloride yielded particles
of increased density. Both microparticle samples showed a final buoyancy
in the Imeron® 300 contrast agent solutions at concentrations of
45-50% contrast agent, regardless of the presence or absence of Tween®
20 surfactant. The conditions for saturation appeared to be dependent
upon the initial pH of the particles, the pH used during the procedure
and the corresponding saturation with methacrylic acid groups in the
particle. At pH below 3.6, constant exchange between protons and cations
was observed. As a result, more beneficial results were shown at pH above
about 3.6 and below about 6.6 to temper the amount of cesium. Within the
preferred range, buoyancy can be varied. At reasonably neutral levels,
based on test at pH of 7.4, the microparticles did not lose their
buoyancy after storage in the contrast agent buffered solution over
night.

EXAMPLE 14

[0123]Further compressibility and mechanical property testing were done on
microspheres in accordance of Samples B and/or C of Example 12. A
pressure test stand which was used for further evaluation is shown in
FIG. 8. An automated syringe plunger 2 having a motor 4 for providing a
variable feed rate of 0 to 250 mm/h and a gear box 6 was further equipped
with a Lorenz pressure transducer 8 capable of measuring forces in the 0
to 500 N range. The syringe plunger 2 was in communication with a syringe
body 10 as shown. The digital output of the transducer was recorded using
a personal computer. The syringe body 10 was filled with 5 ml of a
solution of contrast agent in isotonic phosphate buffer/surfactant
(Tween® 20) solution in a concentration of about 30-32 volume percent
contrast agent. Microparticles were provided to the syringe as well in an
amount of 56 mg dry mass. The syringe contents were then injected through
the microcatheter 12 which was attached to the distal end 14 of the
syringe. The microcatheter had a lumen diameter of 533 μm. The force
needed to push the micropartictes through the catheter into the Petri
dish 16 (shown for receiving microparticle solution) was measured and
recorded as pressure.

[0124]In order to make certain calculations, the following information was
applied as based on typical use of microspheres for injection. Typically
such microspheres have a water content of about 90% such that a vial for
embolization would therefore contain 0.2 mg of embolization particles in
9.8 ml of injection liquid (2 ml of hydrated microparticles in 8 ml
supernatant liquid). Standard preparation procedures include adding 8 ml
of Imeron® 300 contrast agent to the contents of a single vial. This
would provide an equilibrium concentration of contrast agent of 8 ml/(9.8
ml+8 ml)=44.9 volume percent within an injection solution. The solution
is typically drawn up in 1 ml syringes for final delivery. The injection
density thus equals:

ρ=VEmb/VTot=2 ml/18 ml=0.111 Injection agent per volume
fraction.

[0125]The Sample C spheres demonstrated approximately the same equilibrium
water content as typical injectable spheres. To achieve the same
injection density desired for typical surgical procedures, 56 mg of
Sample C microspheres were added to 5 ml of a 31 volume percent contrast
agent solution in isotonic phosphate buffer and surfactant as noted
above.

[0126]The Sample B and C microspheres were evaluated in different
microcatheters of equal lumen diameter at a pH of 7.4. Injections in both
the horizontal and vertical direction were made under different buoyancy
levels and using different swelling levels (based on pH of 6.0 in
contrast to pH 7.4). The results demonstrated that as long as the
diameter of the microspheres was below the internal diameter of the
microcatheter, the microparticles passed through the catheter without
additional frictional force in the same manner as the reference solution.
An increase to about 1.0 to 1.4 kg gravitation force was measured when
the microparticle diameter reached the same dimension as the lumen
diameter. At roughly 20% compression, forces of about 1.5-2.3 kg were
needed to overcome frictional forces within the catheter. Forces greater
than 5 kg were taken as a guideline for moderate to high injection
pressures. When particles are heavier than the injection medium, clogging
was observed when injecting in the vertical position. When injecting the
microparticles in the horizontal position, it was observed that serious
clogging was alleviated and that larger volumes were injectible over
time.

[0127]Injection pressure was further minimized when a lower pH (reduced
swelling) was used in combination with horizontal injection such that the
injection pressures were comparable to the injection media itself. In
addition, injection of Sample C microparticles also exhibited a good
injection pressure pattern at a physiological pH. The catheter entrance
did not clog and each peak in the curve corresponded to either a single
microparticle or number of particles passing through the catheter.

[0128]The results of the various catheter simulation tests shows that the
invention can be used to form injectable microparticles having a density
which substantially matches the density of the injection medium for
injection use. The particles' compressibility can further be such that it
can be injected without forces over more than about 5 kg on the syringe
plunger. The pH of the injection medium can be taken down to about 6 or
injections can be done horizontally to increase the ease of passage of
Sample B and C microparticles through the catheter or injection cannula.
Once within the blood stream, the particles can expand to their original
size in the pH 7.4 environment.

[0129]Additional swelling tests were conducted on the microparticles of
Sample C and it was observed that when ion concentrations were low,
swelling increased. In higher concentrated solutions, swelling decreased.
Continued dilution of the microparticles of Sample C in a buffer solution
led to an increase from 17% to 20% in size of the microparticles. When
mixed into an isotonic phosphate buffer solution, the microparticles
initially increase in size between 83.8 and 97%, wherein in deionized
water, size increases are from about 116.2 to about 136.6%, referring to
the dry particles.

[0130]In further testing to evaluate the compressibility of the
microparticles of Sample C, the syringe pressure test stand of FIG. 8 was
used, however, an optical microscope was used to evaluate the
microparticles as they passed through a progressively narrowed pipette
which was attached to polyethylene tubing connected to the syringe
containing a phosphate buffer solution suspension of microparticles of
Sample C. The pipette narrowed to an inner diameter of 490 μm and the
pipette was mounted to a Petri dish such that the narrowest part was
submerged in phosphate buffer solution to avoid optical distortion and to
collect the liquid ejected from the pipette during measurement. Optical
microscope pictures were taken of the microparticles passing through the
pipette before and during compression. In observing the microparticles,
none of them underwent a fracture, nor did they form debris or coating
delamination after passing through the narrow site. Microparticles which
were chosen to be deliberately too big for the narrow site (for a
compression of about 40%) did not break or rupture, but clogged the
narrow site instead. The maximum compressibility under a reasonable
amount of force on the microparticles while still allowing the
microparticles to pass through the catheter was about 38.7%. Based on
these evaluations, the microparticles according to Sample C demonstrate
properties that would allow particles which are too large to clog the
catheter or cannula rather than break up and cause potential damage to
the patient. The test results provided suggested preferred use parameters
for Sample C microparticles for injection use as shown in Table 2 below.

[0131]Sample C microparticles were further subjected to mechanical and
thermal stress stability testing. Microparticles, after passing through a
Terumo Progreat Tracker catheter were washed with deionized water to
remove residual buffer solution along with contrast agent. They were
dehydrated for 12 h at 60° C. and then transferred to an SEM for
surface analysis. They were compared with particles from the original
batch of microparticles which had undergone the same
hydration/dehydration cycle in milliQ ultrapure water, but which had not
been passed through the catheter. FIGS. 9A and 9B show the surface of the
Sample C microparticles just after the hydration/dehydration cycle and
the film thickness of an exemplary Sample C microparticle, respectively.
SEMs after passing through a catheter at various magnifications (FIGS.
10A, 10B, 10C and 10D) show that the coating did not delaminate (FIG.
10A). Some microparticles did demonstrate some stretching out in the
coating film (FIGS. 10B and 10C). However, a closer magnification as in
FIG. 10D demonstrates that the morphology of the coating layer is still
intact.

[0132]A sterilizer was filled with 2 l of deionized water and 10 vials
each having 56 mg of Sample C microparticles in 3.3 g of solution of
isotonic phosphate buffer/surfactant (Tween® 20) and turned on. The
water boiling point was reached about 15 min. after the start of the
sterilizer, and temperature was held at that point for 3 min. to remove
air by water vapor. The vessel was then sealed shut to raise pressure and
temperature to 125° C. and 1.2 bar pressure. This took
approximately 10 min. The temperature was then maintained for 15 min, and
then the vessel was shut down for a cooling phase. A temperature of
60° C. was reached about 30 min later, after which the vessel was
vented, the samples withdrawn and the vessel shut tightly. A sample vial
was opened, and the supernatant liquid decanted. The microparticles were
washed with deionized water. After dehydration, they were subjected to
measurement using an SEM. The results demonstrated only a small number of
delaminated coatings on the microparticles under such thermal stress (see
FIG. 11A in the strong white contrast portion). The overall percentage of
such microparticles was only about 5 to 10%. Close up, the film
delamination which did occur appears to have occurred along
crystalline-amorphous domain boundaries in the
poly[bis(trifluoroethoxy)phosphazene] coating (see FIG. 11B). Most of the
microparticles showed only minor defects (such as a minor circular patch
being missing), but no damage to the hull of the microparticles (see
FIGS. 11C and 11D).

EXAMPLE 15

[0133]Microparticles were formed in accordance with a preferred embodiment
herein. A deionized water solution of polyvinyl alcohol (PVA) was
prepared using about 23 g of PVA of weight average molecular weight of
about 85,000-124,000, which PVA was about 87-89% hydrolyzed and 1000 g
water. A phosphate buffer solution was prepared using 900 g deionized
water, 4.53 g disodium hydrogen phosphate, 0.26 g sodium dihydrogen
phosphate and 0.056 g ethylenediamine tetraacetic acid (EDTA). Methyl
methacrylate (MMA) monomer was vacuum distilled prior to use.

[0134]Polymerization was carried out in a three-necked, round-bottomed,
2000-ml flask with a KPG mechanical stirring apparatus attached. The
flask was also equipped with a thermometer, reflux condenser and a
pressure release valve with a nitrogen inlet. The polymerization process
further utilized 100 ml of the PVA solution prepared above, 900 ml of the
phosphate buffer solution, 0.65 g of dilauroyl peroxide, 200.2 g
methacrylic acid methyl ester and 2.86 g triethylene glycol
dimethacrylate.

[0135]The PVA and buffer solutions were provided to the reactor flask. The
distilled MMA and triethylene glycol dimethacrylate were introduced,
dilauroyl peroxide then added to the same flask and the components were
agitated to ensure dissolved solids. The reaction flask was flushed with
argon and the stirrer speed set to at 150 rpm to produce particle sizes
of a majority in the range of 300-355 μm. Stirring continued for
approximate 5 minutes. The stirrer was then set to 100 rpm and argon
flushing was discontinued. The reaction flask was then subjected to a
water bath which was heated to 70° C. and held at approximately
that temperature for about 2 hours. The temperature of the bath was then
increased to 73° C. and held for an hour, then the water bath
temperature was raised again to 85° C. and held for another hour.
The stirring and heat were discontinued. The solution was filtered and
the resulting polymethylacrylate microparticles were dried in an oven at
70° C. for about 12 hours. The microparticles were subjected to
sieving and collected in size fractions of from 100-150; 150-200;
200-250; 250-300; 300-355; 355-400; and 400-450 μm with a maximum
yield at 300-355 μm.

[0136]The PMMA microparticles thus formed were then hydrolyzed. A portion
of 100 g 250-300 μm sized microparticles, 150 g potassium hydroxide
and 1400 g of ethylene glycol were added to a 2000 ml flask, reflux
condenser with drying tube connected, and the mixture was heated at
165° C. for 8 hours for full hydrolysis. The mixture was allowed
to cool to room temperature, solution decanted and the microparticles
were washed with deionized water. The procedure was repeated for other
calibrated sizes of microparticles (the following reaction times applied:
300-355 micron particles: 10 hours; 355-400 micron particles: 12 hours
and 400-455 micron particles: 14 hours).

[0137]The microparticles were finally acidified with hydrochloric acid to
a pH of 7.4, and dried in an oven at approximately 70° C.

EXAMPLE 16

[0138]Microparticles formed in accordance with Example 15 were then
esterified in this Example. For esterification surface treatment, 800 g
of dried microparticles from Example 15 were weighed in a 2 L reaction
vessel with a reflux condenser. 250 g thionyl chloride in 1.5 L diethyl
ether were added under stirring. Stirring was continued at room
temperature for 20 hours. The solvent and volatile reactants were removed
by filtration and subsequent vacuum drying. Then 500 g trifluoroethanol
in 1.5 L ether were introduced and the suspension stirred for another 20
hours at room temperature. The particles were finally dried under vacuum.

EXAMPLE 17

[0139]In an alternative surface treatment to Example 16, 800 g dried
microparticles from Example 15 were reacted with 1140 g trifluoroethanol
and 44 g sulfuric acid added as a catalyst. The mixture was stirred for
20 hours at room temperature, filtered and dried under vacuum.

EXAMPLE 18

[0140]800 g of dry PMMA potassium salt microparticles which were partially
esterified with trifluoroethanol as described above in Examples 15-16
were spray coated with poly[bis(trifluoroethoxy)phosphazene] in an MP-1
Precision Coater® fluidized bed coating apparatus (available from
Aeromatic-Fielder AG, Bubendor, Switzerland). The particles were picked
up by an air stream (40-60 m3/h, 55° C. incoming temperature)
and spray coated with poly[bis(trifluoroethoxy)phosphazene] solution
microdroplets from an air-fluid coaxial nozzle. The solution composition
was 0.835 g poly[bis(trifluoroethoxy)phosphazene], 550 g ethyl acetate
and 450 g isopentyl acetate. It was fed through the nozzle's 1.3 mm wide
inner bore at a rate of 10-30 g/min. At the nozzle head, it was atomized
with pressurized air (2.5 bar). The total amount of spray solution (3 kg)
was calculated to coat the particle with a 150 nm thick
poly[bis(trifluoroethoxy)phosphazene] film.

EXAMPLE 19

[0141]The dry potassium salt microparticles of Examples 15-16, which were
partially esterified with trifluoroethanol as described above, were
spray-coated with diluted poly[bis(trifluoroethoxy)phosphazene] solution
in ethyl acetate in a commercially available fluidized bed coating device
(see Example 16). 100 mg of such coated, dried microparticles as well as
100 mg of uncoated, dried PMA potassium salt microparticles which were
partially esterified with trifluoroethanol, were immersed in about 30%
aqueous cesium chloride solution, prepared by dissolving 30.0 g cesium
chloride in 100 ml deionized water. The supernatant liquid was decanted
after 10 min. equilibrium time and the microparticles were washed
thoroughly with deionized water, equilibrated for another 10 min.,
decanted and suspended in 3 ml surfactant free phosphate buffer solution
at a pH of 7.4. Density of the particles in solution was measured for
matching density in a contrast agent solution. To each type of
microparticle was added a contrast agent solution which included a ratio
of 3.5 ml of Imeron® 300 contrast agent (density 1.335 g/ml) and 4 ml
phosphate buffered saline (density 1.009 g/ml). Both hydrogel types
reached buoyancy at levels of 45-50% contrast agent in solution. This
corresponds to an increased density of the microparticles of 1.16 g/ml.

EXAMPLE 20

[0142]Microparticles were formed in accordance with the procedure of
Example 15 with the exception that an exterior barium sulfate coating was
prepared on the microparticles after neutralization of the particles and
the microparticles were not dried after neutralization prior to the
barium sulfate coating step. To prepare the barium sulfate coating, 2500
ml hydrated particles were subjected to 2000 ml of 0.5 M sodium sulfate
(Na2SO4) solution and saturated for 4-12 hours. To the particle
suspension was then slowly added 1950 ml of 0.5 M barium chloride
(BaCl2) solution under stirring at room temperature. After washing
with excess deionized water, the resulting particles in a swollen state
included a barium sulfate powder coated surface. The particles were then
dried and esterified in the manner noted above in Example 16. The
particles were then coated using the fluidized bed process of Example 21
below. The resulting microparticles were externally coated with a
non-adhesive barium sulfate powder. Barium sulfate coatings prepared in
accordance with this invention and procedure are capable of preventing
particle agglomeration during drying and also increase density. The
concentration and ratios of barium sulfate may be varied to provide
different results and a use of an excess of sodium sulfate can minimize
residual barium chloride. The particles formed in accordance with this
example were effectively washed with hot water to minimize excess barium
sulfate powder that may contaminate vials, etc. The barium sulfate works
effectively to prevent adhesion of particles prior to drying to assist in
fluidization of the hydrated microparticles.

EXAMPLE 21

[0143]Fluidized bed coating of barium sulfate powdered beads was performed
using polymethacrylate beads with a surface layer of barium sulfate
formed in accordance with Example 20 but an excess of barium chloride was
used such that barium ions diffused inside the core and formed a
precipitate inside the hydrogel core.

[0144]In preparing the particles, the same procedure for barium sulfate
coated particles set forth in Example 20 was repeated with the exception
that the order of addition was reversed. Thus, 2500 ml hydrated
microparticles were suspended in 2500 ml deionized water and slowly, 5
mol % (200 ml) of a 0.5 M (BaCl2) were added slowly under stirring.
The addition was performed within a time period of three minutes to
prevent irreversible barium acrylate formation taking place. The
suspension was then immediately quenched with the double amount (400 ml)
of 0.5 M sodium sulfate (Na2SO4) solution under stirring at
room temperature. Afterwards, the particles were washed three times with
2 L of deionized water each. This procedure precipitated barium sulfate
inside the particles.

[0145]The resulting precipitate was precipitated within the pores of the
hydrogel core and could not be removed by multiple washings with water.
The particles thus formed were found to have a permanent increased
density in contrast to unmodified particles. The density increase was
controllable by the molar amount of barium chloride used. Amounts ranging
from 0-15 mol % of barium chloride were used reproducibly with this
procedure. It was observed during evaluations of this procedure that, if
the time period of addition exceeded 5 minutes, based upon the diffusion
speed of barium chloride within the particles, the outer pores of the
hydrogel core became irreversibly crosslinked, thereby preventing the
barium sulfate precipitate inside from leaching out. This effect was
visible by optical microscopy as the "diffusion front" of the barium
sulfate was clearly visible as a white band inside the particle, whereas
the surface remained clear.

[0146]Both Examples 20 and 21 provided particles having anti-adhesive
properties that tend not to agglomerate during drying processes;
therefore avoiding surface damage. Generally, such an advantage helps
minimize the amount of particles needed for a fluidized bed procedure as
the particles can be fluidized without being completely dried. The
residual water content may be increased up to 1:1 based on dry weight
without agglomeration. The Examples also produce particles with increased
density properties wherein the density change appears to be permanent.

[0147]It should also be understood according to this disclosure that
generally when applying the procedures noted herein, barium sulfate may
be introduced in accordance with the invention in a range of from 0 to
about 100 mol %, and preferably 0 to about 15 mol % to provide particles
that have preferred elasticity, density and mechanical stability
properties.

[0148]The particles formed according to this Example having a barium
sulfate load inside the core were then esterified according to Example 16
and vacuum-dried. 300 g of the dry beads were suspended in 300 g water
which was completely absorbed by the polymethacrylate cores within less
than 1 min while the barium sulfate powdered particle surface appeared
dry and the particles showed no tendency to agglomerate.

[0149]The particles (now 600 g) with 50 weight percent (wt %) water inside
were spray coated with APTMS/poly[bis(trifluoroethoxy)phosphazene] in an
MP-1 Precision Coater® fluidized bed coating apparatus according to
Example 18 with the exception that an additional aminosilane adhesion
promoter was used. The process equipment used was the same as that of
Example 18, but the coating provided included three different layers. A
bottom coating of 3-aminopropyltrimethoxysilane (APTMS) adhesion promoter
was provided upon which was a second coating layer of a mixture of APTMS
and poly[bis(trifluoroethoxy)phosphazene and a third, top coating layer
of poly[bis(trifluoroethoxy)phosphazene. All three spray solutions were
prepared by dissolving the coating material in isopentyl acetate and
ethyl acetate in a 1:1 weight percentage ratio mixture. The first
solution included 35 μl APTMS dissolved in 200 g acetate mixture. The
second solution included 25 μl APTMS and 125 mg
poly[bis(trifluoroethoxy)phosphazene in 150 mg of the acetate mixture and
the third included 50 mg poly[bis(trifluoroethoxy)phosphazene in 60 g of
the acetate mixture. The spray solution quantities and concentrations
refer to the coating of a 300 g batch with 350 μm particles. The
absorbed water evaporated at a rate of 5-10 g/min. The process was
stopped after 30 min when the coating thickness reached 100 nm and the
residual water content was 18.4 wt %.

EXAMPLE 22

[0150]The absorption of organic dyes was tested on microparticles formed
according to Example 15. To 2 ml of phosphate buffered saline solution
containing 1 ml of hydrated beads was provided an amount of 5-10 μl of
the respective dye as a 10 millimolar solution in ethanol. The samples
were incubated for 30-60 minutes at room temperature under gentle shaking
of the vial. Supernatant liquid was discarded and particles were washed
three times with 2 ml of either deionized water, saline or PBS buffer
solution prior to visualization with optical and fluorescence microscopy.
The dyes tested included triphenylmethane derived dyes such as Fluoescein
diacetate and Rhodamin 6G which were evaluated along with carbocyanine
based dyes such as DiI. The triphenylmethane based Fluorecein and
Rhoamine dyes exhibited a specific affinity for the hydrophilic PMMA
hydrogel core through ionic interactions. They were able to easily
withstand the rigorous conditions of repeated washing and steam
sterilization without substantial leaching.

[0151]The carbocyanine dye DiI on the other hand exhibited a high
selectivity for the hydrophobic poly[bis(trifluoroethoxy)phosphazene
shell, without penetrating the hydrophilic PMAA core material. Thus with
the subsequent staining employing the combination of DiI and Fluorescein
diacetate both core and shell could be simultaneously visualized
employing a fluorescence optical microscope. As a result, this procedure
provides a fast, sensitive fluorescence-staining assay for the PMAA
particles that makes core and shell simultaneously visible under
conditions encountered in actual application. This procedure further
enables assessment of the mechanical-elastic stress or damage to the
poly[bis(trifluoroethoxy)phosphazene shell. It further shows the affinity
of certain classes of dyes for the various components of the particle

[0152]Use of these and other dyes may be used to visually identify
selected microspheres, which may be provided and dyed for identification
to indicate certain sizes of microspheres for use in selected clinical or
diagnostic applications. Color-coding may also be used to identify
selected microspheres on the basis of other properties, such as content
of certain therapeutic or diagnostic agents. Applications according to
the present invention may also improve the imaging visualization by
enhancing the particles' buoyancy behavior

[0153]In various embodiments according to the present invention,
microspheres may be produced in calibrated sizes ranging from about 1 to
about 10,000 nanometers in diameter. In one embodiment of the present
invention, microspheres of the present invention may be provided in sizes
of about 40, about 100, about 250, about 400, about 500, about 700, and
about 900 nanometers in diameter, with a visually distinctive color
imparted to each size of microsphere. Other sizes, size ranges, and
calibrated sized microspheres lacking color dye are also included in the
present invention. Microspheres of the present invention may also be
provided in customized sizes and/or with customized colors as specified
by a user for specific clinical diagnostic or therapeutic applications.

EXAMPLE 23

[0154]FIG. 12A shows a bone defect 20 in an exemplary mammalian bone, show
in FIG. 12 A as a lytic defect in a distal radius 10 with the adjacent
ulna 15. An injection cannula 25 is shown entering the bone defect 20.

[0155]FIG. 12B shows the same anatomic site of FIG. 12A, in which a
plurality of microspheres 30 have been injected through an injection
cannula (not shown in FIG. 12B) to fill the bone defect 20.

[0156]In alternate embodiments of the present invention,
poly[bis(trifluoroethoxy)phosphazene may be applied to a bone defect in
other particulate or solid form, or injected or otherwise instilled
therein as a suspension, solution, paste, or gel.

[0158]Experimental results show a better osseous ingrowth due to
poly[bis(trifluoroethoxy)phosphazene, resulting from inhibitation of
adhering connecting tissue. An even better ingrowth can be expected by
the use of growth factors (like BMPs), which can be connected with
poly[bis(trifluoroethoxy)phosphazene.

[0159]It will be appreciated by those possessing ordinary skill in the art
that changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is understood,
therefore, that this invention is not limited to the particular
embodiments disclosed, but it is intended to cover modifications within
the spirit and scope of the present invention as defined by the appended
claims.